CN103845273B - A kind of earthworm peptide of resisting age of skin and its preparation method and application - Google Patents
A kind of earthworm peptide of resisting age of skin and its preparation method and application Download PDFInfo
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- CN103845273B CN103845273B CN201410092717.3A CN201410092717A CN103845273B CN 103845273 B CN103845273 B CN 103845273B CN 201410092717 A CN201410092717 A CN 201410092717A CN 103845273 B CN103845273 B CN 103845273B
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Abstract
Earthworm peptide the invention discloses a kind of resisting age of skin and its preparation method and application, preparation method is using own enzyme or addition protease hydrolytic by earthworm sample liquid, hydrolyzate is retained using membrane filtration and film again, freeze-drying obtains earthworm peptide, and the earthworm peptide for obtaining is applied in cosmetics for resisting age of skin or health products.Through the above way, the method of the present invention is green, simple, the earthworm peptide compatibility for preparing is strong, safe, with obvious antioxidation activity, growth and the propagation of HSF can be effectively facilitated, there is significant facilitation to the secretion synthesis of cellular matrix collagen, with the effect of obvious resisting age of skin, can be applied to the exploitation of cosmetics for resisting age of skin and health products.
Description
Technical field
The present invention relates to biological technical field, the earthworm peptide of more particularly to a kind of resisting age of skin and preparation method thereof and
Using.
Background technology
Peptide is the bioactivator that a class is related to various kinds of cell function in organism, participates in the various complexity of regulation and control human body
Physiology course.The active high, compatibility of peptide is strong, it is safe the features such as, and peptide molecule is again Nano grade, it is easy to quilt
The intestinal absorption of human body, or deep skin is directly entered by skin surface, being quickly absorbed by the body plays a role.Modern pharmacology
Learn research to show, some active peptides not only have antioxidation activity, can also effectively facilitate the growth of HSF
And propagation, especially can be by increasing the activity of SF, effectively facilitate cellular matrix especially collagen
Etc. the synthesis of matrix, significant resisting age of skin effect has been given play to.Therefore, biologically active peptide is cosmetics for resisting age of skin and guarantor
The development of strong conduct industry provides brand-new direction and thinking, and the application in resisting age of skin product is also more and more universal.
Earthworm (Earthworm) is annelida oligochaeta class animal, is the traditional a herb of China, also referred to as
Dragon.Earthworm has an extensive pharmacological action, effect is significant, such as thrombolysis and anti-bolt, it is antitumor, relieving asthma, promote skin repair, antibacterial
Deng effect.Protein is rich in earthworm body, total protein accounts for the 45.90%~68.11% of dry weight, including Lumbrokinase, fat
Albuminoid, caldesmon, earthworm Neo Calcium binding protein and Lumbrokinase etc..At present, the application of earthworm mainly includes tradition
Chinese medicine decoction is made the form that earthworm powder directly takes and is used as medicine, and production is prepared and natural with thrombolysis, anti-bolt and anti-swollen
The high molecular weight protein extract of the effects such as knurl.Additionally, earthworm or a kind of raw material of good production peptide matters, such as have anti-
Control the earthworm peptides products of the aspects such as hypertension and antibacterial application.But have not been reported and active peptide is prepared using earthworm be applied to anti-skin
The exploitation of skin ageing prod, and during preparing polypeptide as development of raw materials using earthworm, still suffer from preparative separation process
It is numerous and diverse, prepare the problems such as target peptide molecular weight is excessive and distribution is excessively wide, and specific aim is not strong.
The content of the invention
The present invention solves the technical problem of providing a kind of earthworm peptide of resisting age of skin and preparation method thereof and should
With, the earthworm peptide there is obvious resisting age of skin to act on, in can apply to cosmetics for resisting age of skin and health products.
In order to solve the above technical problems, one aspect of the present invention is：A kind of ground of resisting age of skin is provided
Imperial peptide, the earthworm peptide is that molecular weight is the earthworm peptide of 300-3000 dalton.
A kind of preparation method of the earthworm peptide of resisting age of skin is provided, including step is：
(1) clean fresh earthworm is blended and obtains earthworm sample liquid, or earthworm dry product is smashed to pieces and is soaked in water obtained earthworm
Earthworm sample liquid；
(2) the earthworm sample liquid is obtained using the enzyme contained in the earthworm sample liquid or the protease hydrolytic of addition
Hydrolyzate rich in earthworm peptide；
(3) ultrafiltration membrance filter by the hydrolyzate through molecular cut off less than or equal to 3000 dalton, filtrate uses again
Molecular weight is that the NF membrane of 300 dalton is retained, and the trapped fluid freeze-drying for obtaining obtains earthworm peptide.
In a preferred embodiment of the present invention, earthworm described in step (1) is Ju Yin sections animals Pheretima aspergillum, popular ring
The blind ring hair Guo of hair earthworm, power simulation, comb, Hubei Pheretimatschiliensis, Zhili Pheretimatschiliensis, different hair Pheretimatschiliensis, Pheretima californica, the back of the body are dark different
One or more in lip earthworm or Eisenia Foetida.
In a preferred embodiment of the present invention, clean earthworm is to clean fresh earthworm described in step (1), is placed in
Obtain within 1-48 hours in water, the ratio of the earthworm dry product smashed to pieces and water is 1g: 0-20ml, and the soak time is 0-48h.
In a preferred embodiment of the present invention, earthworm sample liquid described in step (2) and the volume ratio of the water are 1:
1-10, the condition of the hydrolysis for pH value be 2-10, temperature be 20-70 DEG C, hydrolysis time be 0.5-10h.
A kind of purposes of the earthworm peptide of resisting age of skin in cosmetics for resisting age of skin or health products is provided.
The beneficial effects of the invention are as follows：Earthworm peptide of resisting age of skin of the invention and its preparation method and application, the party
Method is green, simple, and the earthworm peptide compatibility for preparing is strong, safe, with obvious antioxidation activity, can effectively facilitate
The growth of HSF and propagation, have significant facilitation to the secretion synthesis of cellular matrix collagen,
With the effect of obvious resisting age of skin, the exploitation of cosmetics for resisting age of skin and health products is can be applied to.
Specific embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common
All other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model of present invention protection
Enclose.
Embodiment one：
A kind of preparation method of earthworm peptide is provided, including step is：
(1) fresh earthworm is cleaned, is placed in 24h in deionized water or pure water, earthworm is drained internal food residual
Slag, then blended clean earthworm with high speed bruisher, earthworm blends liquid and is fresh earthworm sample liquid；Wherein described fresh earthworm
It is Ju Yin sections animal Pheretima aspergillum (P.aspergillum (E.Perrier)), popular Pheretimatschiliensis (P.vulgaris Chen), prestige
Honest and clean Pheretimatschiliensis (P.guillelmi (Michaelsen)), blind ring hair Guo (P.pectinifera (Michaelsen)) of comb, Hubei
It is Pheretimatschiliensis (P.hupeiensis), Zhili Pheretimatschiliensis (P.tschiliensis), different hair Pheretimatschiliensis (P.diffringens), white
The dark different lip earthworm (Allolobophora caliginosa trapezoides) or red of neck Pheretimatschiliensis (P.californica), the back of the body
One or more in son love victory earthworm (Eisenia foetida) etc..
(2) to 2 times of deionized waters of volume are added in earthworm sample liquid, it is hydrolyzed using itself enzyme, enzymatic hydrolysis condition is：
PH value is that 6.5, temperature is 40 DEG C, hydrolysis 3h, obtains final product the hydrolyzate rich in earthworm peptide.
(3) by hydrolyzate through molecular cut off be 3000 dalton ultrafiltration membrance filters, filtrate use again molecular weight be 300 a left sides
Right NF membrane is retained, trapped fluid freeze-drying, as earthworm peptide.
Embodiment two
A kind of preparation method of earthworm peptide is provided, including step is：
(1) fresh earthworm is cleaned, is placed in 36h in deionized water or pure water, earthworm is drained internal food residual
Slag, then blended clean earthworm with high speed bruisher, earthworm blends liquid and is fresh earthworm sample liquid；
Earthworm dry product is smashed to pieces, is then soaked with 5 times of quality volumes of deionized water or pure water, soak is earthworm
Earthworm sample liquid；
(2) fresh earthworm sample liquid and dried earthworms (earthworm) sample liquid are mixed according to volume ratio 2: 1, are entered using itself enzyme
Water-filling solution, enzymatic hydrolysis condition is：PH value is that 6.5, temperature is 45 DEG C, hydrolysis 6h, obtains final product the hydrolyzate rich in earthworm peptide.
(3) hydrolyzate is 3000 dalton ultrafiltration membrance filters through molecular cut off, and it is 300 or so that filtrate uses molecular weight again
NF membrane retained, trapped fluid freeze-drying, as earthworm peptide.
Embodiment three：
A kind of preparation method of earthworm peptide is provided, including step is：
(1) earthworm dry product is smashed to pieces, is then soaked with 5 times of quality volumes of deionized water or pure water, soak is
It is earthworm sample liquid；
(2) dried earthworms sample liquid 50ml, addition 0.5mg enzyme activity units are the neutral proteinase of 12000U/mg, carry out enzyme
Solve, enzymatic hydrolysis condition is：PH value is that 7.0, temperature is 40 DEG C, hydrolysis 2h, obtains final product the hydrolyzate rich in earthworm peptide.
(3) hydrolyzate is 3000 dalton ultrafiltration membrance filters through molecular cut off, and it is 300 or so that filtrate uses molecular weight again
NF membrane retained, trapped fluid freeze-drying, as earthworm peptide.
Example IV：
The antioxidation activity in vitro experiment of earthworm peptide：
(1) sample solution is prepared：Using deionized water, earthworm peptide is configured to the sample liquid of various concentrations.
(2) preparation of DPPH solution：It is that DPPH is dissolved in absolute ethyl alcohol by 1,1- diphenyl -2- trinitrophenyl-hydrazines, prepares
It is the mother liquor of 1mmol/L into concentration, is deposited in lucifuge in 4 DEG C, it is molten using the DPPH absolute ethyl alcohols for being preceding diluted to 100 μm of ol/L
Liquid.
(3) it is 100 μm of DPPH solution 1mL of ol/L to take 2mL sample liquids and concentration respectively, in successively adding same test tube,
Shake up, 30 minutes are stood at room temperature, in determination sample absorbance under 517nm wavelength.
Clearance rate：K%=[1- (Ai-Aj)/Ac] × 100%, wherein Ai are the suction of 1ml DPPH solution and 2ml sample liquids
Luminosity, Aj is the absorbance of 1ml solvents and 2ml sample liquids, and Ac is the absorbance of 1ml DPPH solution and 2ml solvents.
(4) experimental result：When earthworm peptide concentration is 1mg/ml, the clearance rate to DPPH free radicals is 22.69%, when dense
Degree is improved during to 5mg/ml, and clearance rate is improved to 48.16%, and when concentration is 10mg/ml, clearance rate has reached 92.2%.
Embodiment five：
The antioxidation activity in vitro experiment of earthworm peptide：
(1) sample solution is prepared：Using deionized water, earthworm peptide is configured to the sample liquid of various concentrations.
(2) Tris-HCl4mL that 0.05mmol/L and pH value are 8.2 is added in test tube, water-bath preheating at 25 DEG C
20min, sequentially adds 1mmol/L pyrogallols 0.5mL, testing sample 0.5mL, and 4min is reacted at 25 DEG C, determines the suction of 325nm
Luminosity.The reagent that each pipe is added is as shown in the table：
| Medicine group | Control group | Standard group | Blank group | |
| Tris-HCl(mL) | 4 | 4 | 4 | 4 |
| Pyrogallol (mL) | 0.5 | 0 | 0.5 | 0 |
| Sample liquid (mL) | 0.5 | 0.5 | 0 | 0 |
| H2O(mL) | 0 | 0.5 | 0.5 | 1 |
Clearance rate：K (%)=[1- (A medicines-A controls)/(A standard-A blank)] * 100%
(3) experimental result：When the concentration of earthworm peptide is 2mg/ml, the clearance rate to superoxide anion is 34.31%, when
Concentration is improved during to 20mg/ml, and clearance rate is improved to 96.23%.
Embodiment six：
Earthworm peptide promotes growth and the proliferation experiment of HSF：
(1) normal HSF is inoculated in Tissue Culture Plate with suitable density respectively, uses 0.25% pancreas
Protease digestion cell, is then made into single cell suspension, with every hole 1X10 with the DMEM nutrient solutions containing 10% hyclone5It is individual to connect
Plant in 96 orifice plates or Tissue Culture Dish (30mm), add and contain 10% hyclone and DMEM in high glucose culture medium, synchronization process
Each group cell, in 5%CO2, cultivated in 37 DEG C of incubators.
The medicine of the 0.4mg/ml earthworm peptide solutions of the undressed blank control group of experiment point and addition dissolved in purified water
Group, after being incubated 3~5 days respectively, appropriate MTT is added per hole, continues to cultivate 4h, supernatant discarded.DMSO, shaking, enzyme are added per hole
Mark instrument detects each hole absorbance.Each group cell after equally cultivating 3~5 days, respectively with Trypsin Induced, is collected by centrifugation thin
Born of the same parents, are fixed with the ethanol that volume fraction is 80%.PBS cell 3 times, is separately added into 4 DEG C of iodate containing Triton of preservation
Third pyridine (PI), vibrates into single cell suspension, and lucifuge is stored at room temperature, and with flow cytomery, repeats experiment 3 times.
(2) experimental result：It is 0.35 ± 0.009 to be not added with the OD detected values of earthworm peptide control group, the peptide experiment of addition earthworm
The OD detected values of group are 0.64 ± 0.013.Through flow cytomery, it is 8.6 to be not added with the result of earthworm peptide, is after dosing
15.4.Flow cytometry testing result and MTT testing results all show that the earthworm peptide of 0.4mg/ml concentration can remarkably promote fell
The fibroblastic growth of skin and propagation.
Embodiment seven：
Earthworm peptide promotes HSF's secretion collagen experiment：
(1) tested according to the method for cell culture in embodiment six, experiment point undressed blank control group and
The medicine group of the 0.4mg/ml earthworm peptide solutions of dissolved in purified water is added, is incubated 3~5 days respectively.After culture terminates, 0.5ml is thin
Born of the same parents' medium centrifugal, supernatant uses people I procollagen types c-terminal peptides (PICP) ELISA kit, is grasped according to specification
Make, the secretion situation of detection I procollagen types albumen (Type I procollagen) tests parallel carrying out three times.
(2) experimental result：Based on quantitative result, defined with the content of I procollagen type albumen in blank control group nutrient solution
It is 100%, when addition 0.4mg/ml earthworms peptide solution carries out cell culture, the content of I procollagen type albumen is improved to 152.41
± 4.21%.Compared with blank control group, the I procollagen type protein contents in addition earthworm peptide experimental group substantially increase.
Embodiment eight：
Application of the earthworm peptide in cosmetics for resisting age of skin：
Cosmetic formulations one：By percentage to the quality, (A) caprylic/capric triglyceride 7.00%, ring polydimethylsiloxanes
Alkane 5.00%, cetostearyl alcohol 5.00%, PEG-100 stearates 3.80%, stearine 2.50%, Butyrospermum parkii
(BUTYROSPERMUM PARKII) fruit fat 3.50%, Nipasol 0.10%, (B) stearoyl-glutamic acid sodium 0.20%, go from
Sub- water surplus, methyl hydroxybenzoate 0.20%, (C) xanthans 0.50%, glycerine 6.00%, (D) Bis(hydroxymethyl)imidazolidinyl urea
0.30%th, methyl hydroxybenzoate 0.20%, Nipasol 0.10%, earthworm peptide 1.00%.
Preparation method：It is completely dissolved it to 80-85 ° A, B heat phase respectively, then A is added to B phases and carries out homogeneous,
Well mixed C is added to simultaneously, homogeneous 5-10min, homogeneous is incubated 30min while stirring in water-bath after terminating, so
After D is added to when being cooled to less than 50 °, wherein dissolve earthworm peptide with a small amount of sterile deionized water being added again into solution, stir
Uniformly, room temperature is cooled to obtain final product.
Cosmetic formulations two：By percentage to the quality, dipotassium hydrogen phosphate 0.18%, potassium dihydrogen phosphate 0.13%, glycerine
2.00%th, 1,3-BDO 1.50%, double PEG-18 methyl polyether silicones 3.00%, rilanit special 1.00%, benzene oxygen second
Alcohol 0.30%, methylisothiazolinone 0.10%, earthworm peptide 0.60%, sterile deionized water surplus.
Preparation method：Above-mentioned raw materials are added in sterile deionized water successively, are stirred while adding, until transparent be
.
Cosmetic formulations three：By percentage to the quality, (A) polyoxyethylene (21) tristearin alcohol ether 1.20%, PEG-100 is stearic
Acid esters 1.50%, stearine 1.00%, isopropyl isostearate 3.50%, caprylic/capric triglyceride 5.00%, whale
Wax ester 2.00%, hexadecanol 1.00%, (B) 1,3-BDO 3.00%, acrylic acid dimethyltaurine ammonium/vp copolymers
0.50%, (C) Dimethicone Crosspolymer 1.00%, dimethyl silicone polymer 3.00%, (D) acrylamide/the third
Enoyl- dimethyltaurine sodium polymer/isohexadecane/polysorbate80 0.90%, (D) glycerine 3.00%, deionized water
Surplus, (F) citric acid 0.01%, sodium citrate 0.05%, NaOH 0.04%, (G) Phenoxyethanol 0.80%, earthworm peptide
0.90%.
Preparation method：A phases are heated separately to 80-90 ° with E phases, homogeneous is carried out during A is then added B, while by B, C
Added with D, homogeneous 5-10min is incubated 30min while stirring after the completion of homogeneous, the water for respectively preparing F phases when being cooled to 50 °
Solution is added with the aqueous solution that G phases are prepared, and is stirred, and is cooled to room temperature and is obtained final product.
Cosmetic formulations four：By percentage to the quality, (A) EDTA-2Na0.05%, PEG-4000 1.20%, go from
Sub- water surplus, glycerine 5.00%, (B) 1,3-BDO 6.00%, Sodium Polyacrylate 0.80%, Carbomer 0.60%, (C)
KOH0.20%, (D) Phenoxyethanol 0.70%, earthworm peptide 0.50%.
Preparation method：By A heat phases to 85-90 °, then well mixed B is added to and carries out homogeneous 5-10min,
Cooling, the aqueous solution that C phases are prepared when temperature is 60 °, added and stirred, and when being cooled to less than 50 °, adds D phases water-soluble
Liquid, stirs, and is cooled to room temperature and obtains final product.
Embodiment nine：
Application of the earthworm peptide in anti-aging health products：
Capsule formula：By percentage to the quality, microcrystalline cellulose 79.98%, earthworm peptide 20.00%, phosphatidase 0 .02%.
Preparation method：Above-mentioned each material is fully milled respectively, crosses 100 eye mesh screens, is then proportionally added, and is carried out
Fully mix, filling capsule is obtained final product.
Oral liquid prescription：By percentage to the quality, earthworm peptide 1.20%, dried fruit juice 3.00%, citric acid 1.00%, trichlorine
Sucrose 0.05%, essence 0.02%, deionized water surplus.
Preparation method：Above-mentioned each material is proportionally sequentially added in deionized water, after stirring, through 300 eye mesh screens
Obtained final product after filtering.
Embodiments of the invention are the foregoing is only, the scope of the claims of the invention is not thereby limited, it is every to utilize this hair
Equivalent structure or equivalent flow conversion that bright description is made, or directly or indirectly it is used in other related technology necks
Domain, is included within the scope of the present invention.
Claims (4)
1. the earthworm peptide of a kind of resisting age of skin, it is characterised in that the earthworm peptide is that molecular weight is 300-3000 dalton
Earthworm peptide；
The preparation method of the earthworm peptide of the resisting age of skin is including step：
(1) clean fresh earthworm is blended and obtains earthworm sample liquid, or earthworm dry product is smashed to pieces and is soaked in water obtained earthworm
Sample liquid；
(2) the earthworm sample liquid obtains richness using the enzyme contained in the earthworm sample liquid or the protease hydrolytic of addition
Hydrolyzate containing earthworm peptide；
(3) ultrafiltration membrance filter by the hydrolyzate through molecular cut off less than or equal to 3000 dalton, filtrate uses molecule again
Measure as the NF membrane of 300 dalton is retained, the trapped fluid freeze-drying for obtaining obtains earthworm peptide；
Purposes of the earthworm peptide in cosmetics for resisting age of skin or health products.
2. the earthworm peptide of resisting age of skin according to claim 1, it is characterised in that earthworm is described in step (1)
Ju Yin sections animals Pheretima aspergillum, popular Pheretimatschiliensis, power simulation, the blind Pheretimatschiliensis of comb, Hubei Pheretimatschiliensis, Zhili ring hair
One or more in the dark different lip earthworm of earthworm, different hair Pheretimatschiliensis, Pheretima californica, the back of the body or Eisenia Foetida.
3. the earthworm peptide of resisting age of skin according to claim 1, it is characterised in that clean earthworm described in step (1)
Earthworm is to clean fresh earthworm, is placed in water 1-48 hours and obtains, and the ratio of the earthworm dry product smashed to pieces and water is 1g:
0-20ml, the soak time is 0-48h.
4. the earthworm peptide of the resisting age of skin according to claim 1, it is characterised in that earthworm described in step (2)
The volume ratio of earthworm sample liquid and water is 1: 1-10, and the condition of the hydrolysis is that 2-10, temperature are 20-70 for pH values
DEG C, hydrolysis time be 0.5-10h.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104887541A (en) * | 2015-02-27 | 2015-09-09 | 济宁华能制药厂有限公司 | Cosmetic containing earthworm active protein |
| CN106722934B (en) * | 2016-11-25 | 2020-10-30 | 济南睿呋康生物科技有限公司 | Anti-aging health food containing earthworm and clam worm peptide-containing extract |
| CN107927764A (en) * | 2017-11-20 | 2018-04-20 | 黑龙江美丽石岛科技开发有限公司 | A kind of health food containing sea cucumber extract and earthworm albumen powder and preparation method thereof |
| CN109293737B (en) * | 2018-09-27 | 2020-10-27 | 华南理工大学 | Tetrapeptide for resisting skin aging and application thereof |
| CN110025559B (en) * | 2019-05-22 | 2021-11-19 | 重庆工商大学 | Earthworm extract, preparation method thereof and skin care product |
| CN110339118B (en) * | 2019-08-19 | 2022-08-16 | 广州明辉化妆品有限公司 | Whitening cream with whitening and moisturizing effects |
| CN111905095B (en) * | 2020-06-30 | 2024-01-05 | 广州市科能化妆品科研有限公司 | Earthworm polypeptide and preparation method and application thereof |
| CN113845565B (en) * | 2021-10-20 | 2023-05-02 | 华中农业大学 | Lumbricus bioactive small peptide, and preparation method and application thereof |
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| CN101020714A (en) * | 2007-03-12 | 2007-08-22 | 唐胜建 | Earthworm protein with function of lowering blood pressure and its prepn process |
| CN102920738A (en) * | 2012-12-04 | 2013-02-13 | 周维官 | Production method of active earthworm protein |
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| KR890004724A (en) * | 1987-09-17 | 1989-05-09 | 히사시 미하라 | New protease preparations |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020714A (en) * | 2007-03-12 | 2007-08-22 | 唐胜建 | Earthworm protein with function of lowering blood pressure and its prepn process |
| CN102920738A (en) * | 2012-12-04 | 2013-02-13 | 周维官 | Production method of active earthworm protein |
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Effective date of registration: 20201104 Address after: Beside Taojing Road, Xinzhuang section, 227 provincial road, Changshu City, Suzhou City, Jiangsu Province Patentee after: Carlyle Cosmetics Co., Ltd Patentee after: TSINGHUA University Address before: 215500 long nilfisk bio Industrial Park, Suzhou, Jiangsu, Changshu Patentee before: JIANGSU LONGRICH BIOTECH Co.,Ltd. Patentee before: TSINGHUA University |
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